Aspergillus terreus HBJ5-32 and application thereof in extracting total flavonoids from chrysanthemum morifolium

Abstract

The application discloses Aspergillus terreus HBJ5-32 and application thereof in extracting total flavones from camellia sinensis, and the fermentation liquor obtained by culturing the Aspergillus terreus HBJ5-32 is filtered, and the collected filtrate is crude enzyme liquor; the crude enzyme liquor is mixed with camellia sinensis powder, and enzymolysis is carried out at 35-40 DEG C for 4-6 h; then, anhydrous ethanol is added to the enzymolysis system, and ultrasonic extraction is carried out; suction filtration is carried out, and total flavone extract liquor is obtained; the total flavone extract liquor is evaporated under reduced pressure, dissolved in anhydrous ethanol, concentrated and vacuum dried, and total flavone extract is obtained. The camellia sinensis microbial enzymolysis method provided by the application can increase the yield of total flavones extracted from camellia sinensis by 26.8% compared with the direct ethanol ultrasonic extraction method.

Description

(I) Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a strain of Aspergillus terreus HBJ5-32 and its application in the extraction of total flavonoids from Hangzhou white chrysanthemum. (II) Background Technology

[0002] Hangzhou white chrysanthemum, also known as small white chrysanthemum, is the capitulum of the chrysanthemum plant (Chrysanthemum morifolium Ramat.) in the Asteraceae family. When brewed as tea, Hangzhou white chrysanthemum has a rich aroma and a sweet taste, and is believed to have effects such as dispelling wind and clearing heat, calming the liver and improving eyesight, and clearing heat and detoxifying. During the Ming Dynasty, Hangzhou Prefecture designated high-quality small white chrysanthemums from Tongxiang as tribute, hence the name Hangzhou white chrysanthemum, also known as "Hangzhou tribute chrysanthemum," and it was as famous as "Longjing tea." Hangzhou white chrysanthemum is slightly cold in nature, bitter and sweet in taste, and enters the lung and liver meridians. It is used to treat wind-heat colds, headaches and dizziness, red and swollen eyes, blurred vision, carbuncles, and boils. The active ingredients of Hangzhou white chrysanthemum mainly include flavonoids, polysaccharides, volatile oils, triterpenoids, steroids, phenols, and trace elements. Among them, flavonoids are one of the main bioactive components of Hangzhou white chrysanthemum, which have antibacterial, antiviral, antioxidant, lipid-lowering, and antitumor effects. They have broad application prospects in the fields of adjuvant drugs, health foods, and fruit and vegetable preservation.

[0003] Currently, there are numerous reports on methods for extracting total flavonoids from chrysanthemum, primarily including hot water extraction, alkaline water extraction, organic solvent extraction, and supercritical CO2 extraction. Based on the first three methods, there are further methods to enhance extraction, such as ultrasound, microwave, pressure, and enzymatic hydrolysis. Each extraction method has its own advantages and disadvantages, resulting in significant differences in total flavonoid yield. For example, hot water extraction is low-cost but yields a low total flavonoid rate, and the extract contains a large amount of polysaccharides and proteins. Organic solvent extraction often uses ethanol, which offers a high yield but requires a large amount of ethanol. Ultrasonic or microwave-assisted extraction, based on water or ethanol extraction, adds ultrasound or microwave treatment to promote the dissolution of total flavonoids, thus improving the yield. Supercritical CO2 extraction yields high purity of total flavonoids, but the yield is often low, and the equipment requirements are relatively high. Enzyme-assisted extraction offers mild extraction conditions and minimizes the inactivation of active substances, but its effect on improving the yield is often not ideal, and commercially available pure enzymes are expensive. Compared with the above-mentioned extraction methods for total flavonoids from Hangzhou white chrysanthemum, ultrasound-assisted ethanol extraction has certain advantages, including higher extraction yield and product purity, the ability to recycle and reuse ethanol, and lower cost of ultrasound.

[0004] Numerous studies have reported the application of enzymatic hydrolysis technology to the extraction of total flavonoids from Hangzhou white chrysanthemum, demonstrating its ability to significantly improve the extraction yield. For instance, Tian Xiulan used cellulase-assisted ethanol extraction of total flavonoids from Hangzhou white chrysanthemum, increasing the yield by 12% [Tian Xiulan. Application of cellulase in the extraction of total flavonoids from Hangzhou white chrysanthemum. Journal of Qiqihar Medical College, 2005, 26(5):542]; Xiang Leiwen used cellulase-assisted hot water extraction of total flavonoids, increasing the yield by 19.2% [Xiang Leiwen, Chen Wentao. Optimization of cellulase extraction process for total flavonoids from Hangzhou white chrysanthemum. Chemical Engineering & Equipment, 2009, (5):26-29]. The cell wall of plants consists of substances such as cellulose, hemicellulose, lignin, pectin, and protein. Using cellulase alone often fails to achieve ideal results. To improve the efficiency of enzymatic extraction, many studies have adopted a compound enzyme method, which involves using two or more enzymes such as cellulase, pectinase, and protease simultaneously. While this can significantly improve the product extraction yield, the use of multiple enzymes undoubtedly increases the extraction cost.

[0005] Microorganisms have a powerful ability to produce enzymes, especially some actinomycetes and molds, which can produce a variety of hydrolytic enzymes that break down plant tissues, including cellulase, hemicellulase, ligninase, pectinase, and protease. Therefore, if a certain microorganism is cultured under suitable conditions, the fermentation broth will contain a large number of hydrolytic enzymes. Using the fermentation broth (crude enzyme solution) to directly hydrolyze plant raw materials, these enzymes can synergistically break down cell wall components, thereby facilitating the release of active ingredients and significantly improving the extraction rate. Moreover, using unpurified crude enzyme solution reduces enzyme costs.

[0006] To improve the extraction yield of total flavonoids from Hangzhou white chrysanthemum, this invention uses crude enzyme solution prepared by microbial fermentation to enzymatically hydrolyze Hangzhou white chrysanthemum, which can significantly increase the extraction yield of total flavonoids from Hangzhou white chrysanthemum. (III) Summary of the Invention

[0007] The purpose of this invention is to provide a novel microbial strain—Aspergillus terreus HBJ5-32—and its application in the extraction of total flavonoids from Hangzhou white chrysanthemum. Enzymatic hydrolysis of the crude enzyme solution prepared by fermentation of Hangzhou white chrysanthemum with this strain can significantly improve the extraction yield of total flavonoids.

[0008] The technical solution adopted in this invention is:

[0009] This invention provides a novel microbial strain—Aspergillus terreus HBJ5-32, deposited at the Guangdong Provincial Center for Microbial Culture Collection, accession number: GDMCC No:62493, deposit date: May 18, 2022, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province; postcode: 510070.

[0010] The *Aspergillus terreus* HBJ5-32 described in this invention is an excellent strain isolated from microbial enrichment cultures of *Chrysanthemum indicum*, obtained through screening and mutagenesis. The colony morphology of *Aspergillus terreus* HBJ5-32 is as follows: On potato dextrose agar (PDA) plates, after 24 hours of cultivation at 28°C, radial grayish-yellow hyphae grow along both sides of the inoculation line; after 48 hours of cultivation, the colonies are thin, yellowish-brown, and contain numerous powdery spores on the surface; the conidiophores are short, the apical sac is hemispherical, the pedicels are double-layered, and the bases of the conidiophores are densely packed, covering the upper two-thirds of the apical sac, exhibiting a fan-shaped appearance under an optical microscope; the conidia are spherical to nearly spherical, with smooth walls, and a diameter of approximately 2.0–2.5 μm. A photograph of *Aspergillus terreus* HBJ5-32 colonies after 2 days of cultivation on PDA plates at 28°C is shown below. Figure 1 .

[0011] The nucleotide sequence of the ribosomal DNA internal transcribed spacer (rDNA-ITS) of Aspergillus terreus HBJ5-32 is shown in SEQ ID NO.1.

[0012] The present invention also provides an application of Aspergillus terrestris HBJ5-32 in the extraction of total flavonoids from Hangzhou white chrysanthemum. The method of application is as follows: (1) the fermentation broth obtained by culturing Aspergillus terrestris HBJ5-32 is filtered, and the collected filtrate is the crude enzyme solution; (2) the crude enzyme solution is mixed with Hangzhou white chrysanthemum powder, and enzymatically hydrolyzed at 35–40℃ for 4–6 h. Then anhydrous ethanol is added to the enzymatic hydrolysis system and ultrasonic extraction is performed. The mixture is filtered to obtain the total flavonoid extract; (3) the total flavonoid extract is evaporated under reduced pressure, dissolved in anhydrous ethanol, concentrated and vacuum dried to obtain the total flavonoid extract.

[0013] Further, the method for preparing the crude enzyme solution in step (1) is as follows: Aspergillus terrestris HBJ5-32 spores are inoculated into the enzyme-producing medium and cultured at 30℃ and 200–250 r / min for 60–72 h with shaking. The fermentation broth is filtered, and the filtrate is the crude enzyme solution. The final concentration composition of the enzyme-producing medium is: wheat bran 30–40 g / L, (NH4)2SO4 5–6 g / L, KH2PO4 3–5 g / L, MgSO4·7H2O 0.5–1.0 g / L, CaCl2 0.3–0.5 g / L, FeSO4·7H2O 0.1–0.2 g / L, the solvent is tap water, and the pH is 6.0–6.5.

[0014] Furthermore, the preferred composition of the enzyme-producing culture medium is: 40 g / L wheat bran, 5 g / L (NH4)2SO4, 3 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.5 g / L CaCl2, 0.1 g / L FeSO4·7H2O, with tap water as the solvent and pH 6.0.

[0015] Before the enzyme-producing culture of Aspergillus terreus HBJ5-32 described in this invention, it needs to be cultured on a plate medium to produce spores. Then, the spores are suspended in physiological saline to obtain Aspergillus terreus HBJ5-32 spore solution, which is then inoculated into the enzyme-producing medium at a volume fraction of 6%–8% for further culture. The specific enzyme-producing culture method is as follows:

[0016] ① Spore suspension preparation: Aspergillus terreus HBJ5-32 was inoculated onto potato dextrose agar (PDA) plates and cultured at 28–30℃ for 48–60 h to obtain plate cultures; sterile physiological saline was added to the plate cultures, and the spores were suspended by stirring with an inoculation loop to obtain Aspergillus terreus HBJ5-32 spore suspension; the final concentration composition of the PDA plate culture medium was: 200 g / L potato (cut into small pieces, boiled in water for 20 min, and the residue was removed and the liquid was retained), 20 g / L glucose, 20 g / L agar, with tap water as the solvent and natural pH (6.5 as measured).

[0017] ② Enzyme production culture: The activated Aspergillus terreus HBJ5-32 spore liquid was inoculated into the enzyme production medium at a volume fraction of 6%–8% and cultured at 30℃ and 200–250 r / min for 60–72 h to obtain a fermentation broth with a dry cell concentration of 4.34–4.79 g / L and a β-glucosidase activity of 35.6–38.8 U / mL.

[0018] Furthermore, the volume of crude enzyme solution used in step (2) is 4–8 mL / g based on the mass of Hangzhou white chrysanthemum powder (i.e., the material-to-enzyme ratio is 1:4–1:8). The Hangzhou white chrysanthemum powder is fine powder obtained by drying Hangzhou white chrysanthemum at 85℃ for 2 hours and then pulverizing it through a 20-mesh sieve. The volume of anhydrous ethanol used is 3–4 times the volume of crude enzyme solution.

[0019] Further, the preparation method of the total flavonoid extract in step (2) is as follows: after the enzymatic hydrolysis of the Hangzhou white chrysanthemum is completed, add 3-4 times the volume of the crude enzyme solution of anhydrous ethanol to the enzymatic hydrolysis system, stir evenly, and then place it in a water bath at 50-60℃ for 2-3 hours for extraction, and then transfer it to an ultrasonic cleaner at 50-60℃ and 100-200W for 30-60 minutes for extraction; after the ultrasonic ethanol extraction is completed, filter while hot to obtain the total flavonoid extract.

[0020] Further, the method for recovering total flavonoids from the total flavonoid extract in step (3) is as follows: the total flavonoid extract is distilled at 45℃ and –0.1MPa until no liquid flows out, and anhydrous ethanol with a volume of 5–10 mL / g based on the mass of the raw material, Hangzhou white chrysanthemum, is added. After thorough shaking, it is centrifuged at 5000–8000 r / min for 5–10 min. The supernatant is distilled at 45℃ and –0.1MPa to 1 / 10 of the original ethanol volume, transferred to a clean petri dish, and vacuum dried at 50℃ and –0.1MPa to obtain the total flavonoid extract.

[0021] Compared with existing technologies, the beneficial effects of this invention are mainly reflected in the following: before the ultrasonic extraction of total flavonoids from Hangzhou white chrysanthemum with ethanol, a crude enzyme solution prepared by microbial fermentation is added for enzymatic hydrolysis. The enzyme-producing microorganism used, *Aspergillus terreus* HBJ5-32, was specifically screened to hydrolyze Hangzhou white chrysanthemum plant tissue. The fermentation broth cultured with this enzyme contains a variety of hydrolytic enzymes, which can synergistically hydrolyze substances such as cellulose and pectin in Hangzhou white chrysanthemum, causing cell wall breakage and facilitating the dissolution of flavonoids, thus significantly improving the extraction yield of total flavonoids. The microbial enzymatic hydrolysis method for Hangzhou white chrysanthemum provided by this invention can increase the yield of total flavonoids extracted from Hangzhou white chrysanthemum by 26.8% compared with the direct ultrasonic extraction method with ethanol. (iv) Description of the attached drawings

[0022] Figure 1 This is a colony photo of Aspergillus terreus HBJ5-32 after being cultured on a PDA at 28°C for 48 hours.

[0023] Figure 2 The standard curve for the spectrophotometric determination of total flavonoids is provided.

[0024] Figure 3 The standard curve for spectrophotometric determination of nitrophenol is provided. (V) Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0026] The Hangzhou white chrysanthemum described in this invention is the capitulum of the chrysanthemum (Chrysanthemum morifolium Ramat.), a plant of the Asteraceae family, with a diameter of 2.5-4.0 cm, and originating from Tongxiang City, Zhejiang Province. The Hangzhou white chrysanthemum powder is a fine powder obtained by drying Hangzhou white chrysanthemum at 85℃ for 2 hours, then pulverizing it and passing it through a 20-mesh sieve.

[0027] Example 1: Isolation and screening of fermentation strains

[0028] The microbial strains that produce enzymes for fermentation and enzymatic hydrolysis of Hangzhou white chrysanthemum were isolated and screened according to the following steps:

[0029] (1) Add 10g of Hangzhou white chrysanthemum powder to a 250-mL Erlenmeyer flask, add 15mL of sterile physiological saline, stir well, and incubate at 28℃ for 72h. Dilute the enriched culture contaminated with mold with sterile physiological saline at 1×10⁻⁶. -5 1×10 -6 1×10 -7 After dilution, 0.1 mL of the diluted solution was spread onto potato dextrose agar (PDA) plates and incubated at 28°C for 48 h. Mold colonies with different colors and morphologies were picked and transferred to fresh PDA plates and incubated at 28°C for 60 h to obtain 6 pure culture strains. The strain numbers are shown in Table 1.

[0030] (2) 10 mL of sterile physiological saline was added to each of the six strains of fresh plate culture, and the spores were suspended by stirring with an inoculation loop to obtain the spore solution of each strain.

[0031] (3) Take 3 mL of the spore liquid of each strain prepared in step (2) and inoculate it into 50 mL of enzyme production medium (the inoculation amount is 6% by volume). After culturing for 72 h under shaking conditions at 30℃ and 200 r / min, filter all the fermentation broth with a Buchner funnel. The collected filtrate is the crude enzyme solution.

[0032] (4) Add 0.5g of Hangzhou white chrysanthemum powder to each of the six 50-mL centrifuge tubes, add 4mL of crude enzyme solution of each strain prepared in step (3) (material-enzyme ratio of 1:8), stir evenly, and then enzymatically hydrolyze at 35℃ for 6h to obtain Hangzhou white chrysanthemum hydrolysate.

[0033] (5) In step (4), 12 mL of anhydrous ethanol (3 times the volume of crude enzyme solution, the volume concentration of ethanol in the system is 75%) was added to all the enzymatic hydrolysates of the Hangzhou white chrysanthemum after enzymatic hydrolysis by each strain. After shaking well, the mixture was placed in a 50℃ water bath for 2 hours and then transferred to an ultrasonic cleaner at 50℃. The mixture was ultrasonically extracted at 100W for 60 minutes. The mixture was filtered through a Buchner funnel while hot, and the filtrate was collected. The total flavonoid content was determined by spectrophotometry.

[0034] Under the same conditions, 4 mL of uninoculated enzyme-producing medium was used as a control instead of crude enzyme solution; 4 mL of cellulase phosphate buffer (pH 6.0, 0.2 mol / L) with an activity of 2000 U / mL was used as a control instead of crude enzyme solution for cellulase digestion. The total flavonoid extraction yields of chrysanthemum and controls prepared by fermentation of different strains are shown in Table 1.

[0035] Table 1. Total flavonoid extraction yield of chrysanthemum from crude enzyme solutions prepared by fermentation of different strains and control.

[0036]

[0037] As shown in Table 1, the total flavonoid extraction rate of Hangzhou white chrysanthemum after enzymatic hydrolysis with crude enzyme solution prepared by HBJ5 strain was 7.47%, which was 12.3% higher than the 6.65% of the unhydrolyzed control. Treatment of Hangzhou white chrysanthemum with cellulase also improved the total flavonoid extraction rate by 5.71% compared to the unhydrolyzed control, but this was far less than that of the crude enzyme solution prepared by HBJ5 strain fermentation. Enzymatic hydrolysis of Hangzhou white chrysanthemum with crude enzyme solution from some microbial strains did not improve the total flavonoid extraction rate, and even decreased it. These results indicate that the microbial strains used for enzymatic hydrolysis of Hangzhou white chrysanthemum to improve the total flavonoid extraction rate are selective; they must not only produce enzymes to hydrolyze the cell wall components of Hangzhou white chrysanthemum, but also not produce enzymes to degrade flavonoids. In this invention, HBJ5 strain was selected as the enzyme-producing strain for improving the total flavonoid extraction rate of Hangzhou white chrysanthemum.

[0038] The PDA plate culture medium is prepared according to the following composition and method: Wash and peel potatoes, cut them into small cubes with a side length of about 1cm, weigh 200g, add 1000mL of tap water, boil for 20min, filter through 4 layers of gauze to remove residue, add the filtrate to 1000mL, add 20g of glucose and 20g of agar, set the pH to natural (actually measured to be around 6.5), heat until the agar dissolves, dispense into Erlenmeyer flasks, sterilize by high pressure steam at 121℃ for 20min, and before solidification, pour into 9cm diameter sterile petri dishes, 15-20mL per dish.

[0039] The final concentration composition and preparation method of the enzyme-producing culture medium are as follows: 40 g / L wheat bran, 5 g / L (NH4)2SO4, 3 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.5 g / L CaCl2, 0.1 g / L FeSO4·7H2O, with tap water as the solvent and pH 6.0. 50 mL of the enzyme-producing culture medium is placed in a 250 mL Erlenmeyer flask, sealed with 8 layers of gauze, and sterilized by autoclaving at 121°C for 20 min.

[0040] The total flavonoid content was determined by spectrophotometry. The specific method was as follows: 2.5 mL of total flavonoid extract (adjust the sample volume appropriately according to the total flavonoid concentration in the sample, and control the determination of A...). 510 = Between 0.2 and 0.8, the crude total flavonoid extract was dissolved in 60% ethanol aqueous solution. In a 10-mL graduated test tube, 0.4 mL of 5% sodium nitrite (NaNO2) aqueous solution was added, and the mixture was allowed to stand for 6 min. Then, 0.6 mL of 10% aluminum nitrate [Al(NO3)3] aqueous solution was added, and the mixture was allowed to stand for 6 min. Then, 2 mL of 20% sodium hydroxide (NaOH) aqueous solution was added, and the volume was adjusted to 10 mL with 60% ethanol aqueous solution. The mixture was shaken well and allowed to stand for 15 min. The absorbance at 510 nm was measured using a spectrophotometer. 510The total flavonoid concentration in the sample is calculated from the rutin standard curve, and then the total flavonoid mass is obtained by multiplying the concentration by the volume of the sample.

[0041] Construction of the rutin standard curve: Prepare a 0.25 g / L rutin standard solution using a 60% ethanol aqueous solution. Take 0, 0.5, 1.0, 1.5, 2.0, and 2.5 mL of the rutin standard solution into 10-mL graduated test tubes, respectively, and bring the volume to 2.5 mL with 60% ethanol aqueous solution. Add 0.4 mL of 5% sodium nitrite aqueous solution, let stand for 6 min, add 0.6 mL of 10% aluminum nitrate aqueous solution, let stand for 6 min, add 2 mL of 20% sodium hydroxide aqueous solution, and then dilute to 10FeSO4 mL with 60% ethanol aqueous solution. Shake well, let stand for 15 min, and measure the absorbance at 510 nm using a spectrophotometer (A). 510 Plotting rutin concentration on the x-axis, A 510 Plot a standard curve for the ordinate ( Figure 2 ).

[0042] The total flavonoid yield extracted from Hangzhou white chrysanthemum is calculated using the following formula:

[0043]

[0044] Example 2: Mutagenesis and selection of fermentation strain HBJ5

[0045] Mutagenesis breeding was performed on strain HBJ5 to screen for strains with excellent fermentation performance. The specific method is as follows:

[0046] (1) Preparation of spore suspension: After activating strain HBJ5 on PDA agar at 28℃ for 48h, 5mL of sterile physiological saline was added, and the spores were suspended by stirring with an inoculation loop. 1mL of the spore suspension was transferred to an Erlenmeyer flask containing 50mL of sterile physiological saline (with 20–30 glass beads added), and shaken at room temperature for 15min. The spore suspension was filtered to remove mycelia (a small wad of defatted cotton was placed at the bottom of the Erlenmeyer funnel). The spores in the suspension were counted under a microscope using a hemocytometer. The suspension was then diluted appropriately with sterile physiological saline to adjust the spore count to 1.14 × 10⁻⁶. 7 per mL.

[0047] (2) Mutagenesis: Under red light illumination, 1.5 mL of the above spore solution and a sterile paperclip were placed in five 6 cm diameter petri dishes. The petri dishes were placed on magnetic stirrers and irradiated for 1, 2, 3, 4, and 5 mins respectively at a distance of 30 cm from a 15 W UV lamp that had been preheated for 30 min. 0.5 mL of the irradiated spore solution was diluted appropriately and 0.1 mL was transferred to PDA agar plates. The same procedure was performed to dilute and plate the spore solution without UV irradiation as a control to calculate the lethality rate. After inoculation, the PDA plates were wrapped in black cloth, inverted, and incubated at 28 °C for 36 h. The colonies on the plates were counted, and the lethality rate was calculated.

[0048] (3) Screening: Colonies with a lethality rate of over 90% on PDA plates were selected and transferred to fresh PDA plates to obtain 45 strains. 10 mL of sterile physiological saline was added to each strain's fresh plate culture after 60 h of incubation at 30°C, and the spores were suspended by stirring with an inoculation loop to obtain the spore liquid for each strain. 3 mL of the spore liquid from each strain was inoculated into 50 mL of enzyme-producing medium and cultured at 30°C and 200 r / min for 72 h. The fermentation broth was then filtered using a Buchner funnel, and the filtrate was collected. The β-glucosidase activity of the fermentation filtrate for each strain was measured. Fifteen strains with relatively higher enzyme activity than the wild-type strain were selected. Following the method in Example 1, the crude enzyme solution fermented by these 15 strains was used to enzymatically hydrolyze Hangzhou white chrysanthemum. Total flavonoids were extracted by ultrasonic extraction with ethanol. The total flavonoid extraction yield of the crude enzyme solution prepared from the fermentation of mutant strains after further screening is shown in Table 2.

[0049] Table 2. Total flavonoid extraction yield of Hangzhou white chrysanthemum after enzymatic hydrolysis with crude enzyme solution prepared from the fermentation of the double-screened mutant strains.

[0050]

[0051] As shown in Table 2, among the 15 strains screened, strain HBJ5-32 exhibited a β-glucosidase activity of 35.6 U / mL, which was 47.1% higher than the 24.2 U / mL of the wild-type strain HBJ5. The crude enzyme solution prepared by fermentation with this strain yielded a total flavonoid extraction rate of 8.36% after hydrolysis of Hangzhou white chrysanthemum, an increase of 11.9% compared to the 7.47% of the wild-type strain HBJ5 and a 25.7% increase compared to the unhydrolyzed control (6.65%). Therefore, strain HBJ5-32 was selected in this invention as the enzyme-producing strain for improving the total flavonoid extraction rate of Hangzhou white chrysanthemum.

[0052] The method for determining β-glucosidase activity is as follows: Add 0.8 mL of crude enzyme solution and 0.2 mL of 5 mmol / L p-nitrophenyl-β-D-glucopyranoside (pNPG) solution (prepared with pH 6.0 and 0.2 mol / L phosphate buffer) sequentially to a test tube. React at 35°C for 15 min, then add 2 mL of 1 mol / L Na₂CO₃ aqueous solution and shake well to terminate the reaction. Using the same treatment as the boiled and inactivated crude enzyme solution as a reference, measure the absorbance (A) at 400 nm using a spectrophotometer. 400 ). From the concentration of p-nitrophenol (pNP) — A 400 Standard curve ( Figure 3 The concentration of pNP in the reaction system was calculated.

[0053] The definition of β-glucosidase activity unit (U): the amount of enzyme that hydrolyzes pNPG to generate 1 μmol of pNP in 1 min at 35℃ and in a pH 6.0 buffer system is defined as 1 enzyme activity unit.

[0054] Enzyme activity is calculated according to the following formula (1).

[0055]

[0056] In formula (1), V1: total volume of the reaction system; V2: crude enzyme liquid volume; C1: pNP concentration; T: reaction time.

[0057] p-Nitrophenol Concentration—A 400 Preparation of the standard curve: Prepare a 1 mmol / L pNP standard solution using distilled water. Pipette 0.1, 0.2, 0.3, 0.4, and 0.5 mL of the standard solution into 10 mL volumetric flasks, respectively, and dilute to volume with 1 mol / L Na₂CO₃ aqueous solution. Mix well to achieve p-nitrophenol concentrations of 10, 20, 30, 40, and 50 μmol / L in each sample. Using distilled water as a blank, measure the absorbance (A) at 400 nm using a spectrophotometer. 400 ), with the concentration of p-nitrophenol as the x-axis, A 400 Plot the p-nitrophenol concentration – A on the ordinate. 400 Standard curve.

[0058] Example 3: Classification and identification of strain HBJ5-32

[0059] Strawberry strain HBJ5-32 was streaked onto PDA plates and incubated at 28°C for 24 hours. Radial grayish-yellow hyphae grew along both sides of the inoculation line. After 48 hours of incubation, the colonies were thin, yellowish-brown, and contained numerous powdery spores. The conidiophores were short, the apical sac was hemispherical, the pedicels were double-layered, and the bases of the pedicels were densely packed, covering the upper two-thirds of the apical sac, appearing fan-shaped under a light microscope. The conidia were spherical to nearly spherical, with smooth walls, and approximately 2.0–2.5 μm in diameter. A photograph of Aspergillus terreus HBJ5-32 colonies after 2 days of incubation on PDA plates at 28°C is shown below. Figure 1 .

[0060] The rDNA-ITS nucleotide sequence of strain HBJ5-32 was determined as shown in SEQ ID NO.1. This sequence was BLAST-aligned with the typical Aspergillus terreus strain ATCC1012 by NCBI (National Center for Biotechnology Information, https: / / www.ncbi.nlm.nih.gov) and showed 100% homology. Based on the colony morphology characteristics and rDNA-ITS nucleotide sequence alignment of strain HBJ5-32, the biological classification of strain HBJ5-32 can be determined as follows (refer to Mycobank, http: / / www.mycobank.org): Kingdom Fungi, Phylum Ascomycota, Subphylum Pezizomycotina, Class Eurotiomycetes, Subclass Eurotiomycetidae, Order Eurotiales, Family Aspergillaceae, Genus Aspergillus, Genus Aspergillus terreus.

[0061] The ITS region rDNA sequence is as follows:

[0062] .

[0063] In summary, strain HBJ5 was isolated from the microbial enrichment of Hangzhou white inulin. After ultraviolet mutagenesis, strain HBJ5-32, namely Aspergillus terreus HBJ5-32, was obtained through screening. This strain is deposited at the Guangdong Provincial Microbial Culture Collection Center, accession number: GDMCC No:62493, deposit date: May 18, 2022, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province; postcode: 510070.

[0064] Example 4: Extraction of total flavonoids from Hangzhou white chrysanthemum using Aspergillus terrestris HBJ5-32 enzymatic hydrolysis method

[0065] The total flavonoids in Hangzhou white chrysanthemum can be extracted using the enzymatic hydrolysis method of Aspergillus terrestris HBJ5-32, and the following steps can be followed:

[0066] (1) Aspergillus terreus HBJ5-32 spore powder preserved in freeze-dried tubes was inoculated onto fresh PDA plate medium and cultured at 30℃ for 60 h. 10 mL of sterile physiological saline was added to the plate culture, and the spores were suspended by stirring with an inoculation loop to obtain a spore solution of Aspergillus terreus HBJ5-32. The composition and preparation method of the PDA plate medium are the same as in Example 1.

[0067] (2) Inoculate 3 mL of the spore solution prepared in step (1) into 50 mL of enzyme-producing medium (inoculation amount of 6% by volume), and culture at 30℃ and 200 r / min for 72 h with shaking to obtain a fermentation broth with a dry cell concentration of 4.79 g / L. Filter all fermentation broth using a Buchner funnel, and the collected filtrate is the crude enzyme solution. The β-glucosidase activity of the filtrate is 36.7 U / mL. The final concentration composition and preparation method of the enzyme-producing medium are the same as in Example 1.

[0068] (3) Add 5g of Hangzhou white chrysanthemum powder to a 250-mL Erlenmeyer flask, add 40mL of crude enzyme solution prepared in step (2) (material-enzyme ratio of 1:8), stir evenly, and then enzymatically hydrolyze at 35℃ for 6h to obtain Hangzhou white chrysanthemum hydrolysate.

[0069] (4) In step (3), add 120 mL of anhydrous ethanol (3 times the volume of crude enzyme solution, the volume concentration of ethanol in the system is 75%) to all the enzymatic hydrolysate of Hangzhou white chrysanthemum, shake well and place in a 50℃ water bath for 2 h, then transfer to an ultrasonic cleaner at 50℃, ultrasonically extract for 60 min at 100W power, filter with a Buchner funnel to obtain total flavonoid extract.

[0070] (5) The total flavonoid extract prepared in step (4) was distilled under reduced pressure at 45°C and -0.1 MPa until no liquid flowed out. 50 mL of anhydrous ethanol (10 mL / g based on the mass of raw material Hangzhou white chrysanthemum powder) was added, and the mixture was shaken thoroughly and transferred to a centrifuge tube. The mixture was centrifuged at 8000 r / min for 5 min. The supernatant was distilled at 45°C and -0.1 MPa to about 5 mL and transferred to a clean petri dish. The mixture was then dried under vacuum at 50°C and -0.1 MPa to obtain the total flavonoid extract.

[0071] Following the steps above, 0.731g of total flavonoid extract was obtained, with a total flavonoid content of 51.5%, which means 0.376g of total flavonoids were obtained, and the extraction yield was 7.52%.

[0072] Comparative Example 1: Total flavonoids extracted from Hangzhou white chrysanthemum without enzymatic hydrolysis (compared to Example 4)

[0073] (1) 5g of Hangzhou white chrysanthemum powder was placed in a 250-mL Erlenmeyer flask, 160mL of 75% ethanol aqueous solution was added, and the mixture was shaken well and then placed in a 50℃ water bath for 2h. Then it was transferred to an ultrasonic cleaner at 50℃ and ultrasonically extracted for 60min at 100W power. The mixture was filtered through a Buchner funnel to obtain the total flavonoid extract.

[0074] (2) The total flavonoid extract prepared in step (1) was distilled under reduced pressure at 45°C and -0.1 MPa until no liquid flowed out. 50 mL of anhydrous ethanol (10 mL / g based on the mass of raw material Hangzhou white inulin) was added. After thorough shaking, the mixture was transferred to a centrifuge tube and centrifuged at 8000 r / min for 5 min. The supernatant was distilled at 45°C and -0.1 MPa to about 5 mL and transferred to a clean petri dish. The mixture was then dried under vacuum at 50°C and -0.1 MPa to obtain the total flavonoid extract.

[0075] Following the steps above, 0.626g of total flavonoid extract was obtained, with a total flavonoid content of 48.7%, which means 0.305g of total flavonoids was obtained, and the extraction yield was 6.10%.

[0076] Comparing the results of Example 1 and Comparative Example 1, it can be seen that before extracting the total flavonoids from Hangzhou white chrysanthemum, the addition of crude enzyme solution prepared by Aspergillus terrestris fermentation to hydrolyze the total flavonoids increased the extraction yield of total flavonoids from 6.10% to 7.52%, an increase of 23.3%.

[0077] Example 5: Extraction of total flavonoids from Hangzhou white chrysanthemum using Aspergillus terrestris HBJ5-32 enzymatic hydrolysis method

[0078] The total flavonoids in Hangzhou white chrysanthemum can be extracted by enzymatic hydrolysis using Aspergillus terrestris HBJ5-32, following these steps:

[0079] (1) Aspergillus terreus HBJ5-32 PDA colony spores stored at 4℃ were inoculated onto fresh PDA agar plates and cultured at 30℃ for 54 h. 10 mL of sterile physiological saline was added to the agar plate culture, and the spores were suspended by stirring with an inoculation loop to obtain a spore suspension of Aspergillus terreus HBJ5-32. The composition and preparation method of the PDA agar plate culture medium were the same as in Example 1.

[0080] (2) 7 mL of the spore solution prepared in step (1) was inoculated into 100 mL of enzyme-producing medium (inoculation amount of 7% by volume), and cultured at 30℃ and 220 r / min for 66 h to obtain a fermentation broth with a dry cell concentration of 4.67 g / L. All fermentation broth was filtered using a Buchner funnel, and the collected filtrate was the crude enzyme solution. The β-glucosidase activity of the filtrate was 38.1 U / mL. The final concentration composition of the enzyme-producing medium was the same as in Example 1. 100 mL of enzyme-producing medium was placed in a 250-mL Erlenmeyer flask, sealed with 8 layers of gauze, and sterilized by high-pressure steam at 121℃ for 20 min.

[0081] (3) 10g of Hangzhou white chrysanthemum powder was placed in a 500-mL Erlenmeyer flask, and 60mL of crude enzyme solution prepared in step (2) was added (the ratio of material to enzyme was 1:6). After stirring evenly, the mixture was enzymatically hydrolyzed at 40℃ for 5h to obtain Hangzhou white chrysanthemum hydrolysate.

[0082] (4) In step (3), add 240 mL of anhydrous ethanol (4 times the volume of crude enzyme solution, ethanol volume concentration of 80%) to all the enzymatic hydrolysate of Hangzhou white chrysanthemum, shake well and place in a 55℃ water bath for 2.5 h for extraction, then transfer to an ultrasonic cleaner at 55℃ and ultrasonically extract for 45 min at 150 W power. Filter with a Buchner funnel to obtain total flavonoid extract.

[0083] (5) The total flavonoid extract prepared in step (4) was distilled under reduced pressure at 45°C and -0.1 MPa until no liquid flowed out. 75 mL of anhydrous ethanol (7.5 mL / g based on the mass of raw material Hangzhou white chrysanthemum powder) was added, and after thorough shaking, it was transferred to a centrifuge tube and centrifuged at 8000 r / min for 5 min. The supernatant was distilled at 45°C and -0.1 MPa to about 7.5 mL, and transferred to a clean petri dish. It was then dried under vacuum at 50°C and -0.1 MPa to obtain the total flavonoid extract.

[0084] Following the steps above, 1.46g of total flavonoid extract was obtained, with a total flavonoid content of 53.8%, which means 0.785g of total flavonoids were obtained, and the extraction yield was 7.85%.

[0085] Comparative Example 2: Total flavonoids extracted from Hangzhou white chrysanthemum without enzymatic hydrolysis (compared with Example 5)

[0086] (1) 10g of Hangzhou white chrysanthemum powder was placed in a 500-mL Erlenmeyer flask, 300mL of 80% ethanol aqueous solution was added, and the mixture was shaken well and then placed in a 55℃ water bath for 2.5h. Then it was transferred to an ultrasonic cleaner at 55℃ and ultrasonically extracted for 45min at 150W power. The mixture was filtered through a Buchner funnel to obtain the total flavonoid extract.

[0087] (2) The total flavonoid extract prepared in step (1) was distilled under reduced pressure at 45°C and -0.1 MPa until no liquid flowed out. 75 mL of anhydrous ethanol (7.5 mL / g based on the mass of raw material Hangzhou white chrysanthemum powder) was added, and the mixture was shaken thoroughly and transferred to a centrifuge tube. The mixture was centrifuged at 8000 r / min for 5 min. The supernatant was distilled at 45°C and -0.1 MPa to about 7.5 mL and transferred to a clean petri dish. The mixture was then dried under vacuum at 50°C and -0.1 MPa to obtain the total flavonoid extract.

[0088] Following the steps above, 1.23g of total flavonoid extract was obtained, with a total flavonoid content of 50.3%, which means 0.619g of total flavonoids were obtained, and the extraction yield was 6.19%.

[0089] Comparing the results of Example 5 and Comparative Example 2, it can be seen that before extracting the total flavonoids from Hangzhou white chrysanthemum, adding the crude enzyme solution prepared by Aspergillus terreus fermentation HBJ5-32 for enzymatic hydrolysis increased the total flavonoid extraction yield from 6.19% to 7.85%, an increase of 26.8%.

[0090] Example 6: Extraction of total flavonoids from Hangzhou white chrysanthemum using Aspergillus terrestris HBJ5-32 enzymatic hydrolysis method

[0091] The total flavonoids in Hangzhou white chrysanthemum can be extracted by enzymatic hydrolysis using Aspergillus terrestris HBJ5-32, following these steps:

[0092] (1) Aspergillus terreus HBJ5-32 PDA colony spores stored at 4℃ were inoculated onto fresh PDA agar plates and cultured at 30℃ for 48 h. 10 mL of sterile physiological saline was added to the agar plate culture, and the spores were suspended by stirring with an inoculation loop to obtain a spore suspension of Aspergillus terreus HBJ5-32. The composition and preparation method of the PDA agar plate culture medium were the same as in Example 1.

[0093] (2) Inoculate 12 mL of the spore solution prepared in step (1) into 150 mL of enzyme-producing medium (inoculation amount of 8% by volume), and culture at 30℃ and 250 r / min for 60 h to obtain a fermentation broth with a dry cell concentration of 4.34 g / L. Filter all the fermentation broth using a Buchner funnel, and the collected filtrate is the crude enzyme solution. The β-glucosidase activity of the filtrate is 38.8 U / mL. The final concentration composition of the enzyme-producing medium is the same as in Example 1. Fill a 500-mL Erlenmeyer flask with 150 mL of enzyme-producing medium, seal the flask with 8 layers of gauze, and sterilize by autoclaving at 121℃ for 20 min.

[0094] (3) 25g of Hangzhou white chrysanthemum powder was placed in a 1-L Erlenmeyer flask, and 100mL of crude enzyme solution prepared in step (2) was added (the ratio of material to enzyme was 1:4). After stirring evenly, the mixture was enzymatically hydrolyzed at 40℃ for 4h to obtain Hangzhou white chrysanthemum hydrolysate.

[0095] (4) In step (3), add 400 mL of anhydrous ethanol (4 times the volume of crude enzyme solution, ethanol volume concentration of 80%) to all the enzymatic hydrolysate of Hangzhou white chrysanthemum, shake well and place in a 60℃ water bath for 3 h for extraction, then transfer to an ultrasonic cleaner at 60℃ and ultrasonically extract for 30 min at 200W power, filter with a Buchner funnel to obtain total flavonoid extract.

[0096] (5) The total flavonoid extract prepared in step (4) was distilled under reduced pressure at 45°C and -0.1 MPa until no liquid flowed out. 125 mL of anhydrous ethanol (5 mL / g based on the mass of raw material Hangzhou white chrysanthemum powder) was added, and after thorough shaking, it was transferred to a centrifuge tube and centrifuged at 5000 r / min for 10 min. The supernatant was distilled at 45°C and -0.1 MPa to about 12.5 mL, transferred to a clean petri dish, and dried under vacuum at 50°C and -0.1 MPa to obtain the total flavonoid extract.

[0097] Following the steps above, 3.52g of total flavonoid extract was obtained, with a total flavonoid content of 54.3%, which means 1.91g of total flavonoids was obtained, and the extraction yield was 7.64%.

[0098] Comparative Example 3: Total flavonoids extracted from Hangzhou white chrysanthemum without enzymatic hydrolysis (compared to Example 6)

[0099] (1) 25g of Hangzhou white chrysanthemum powder was placed in a 1-L Erlenmeyer flask, 500mL of 80% ethanol aqueous solution was added, and the mixture was shaken well and then placed in a 60℃ water bath for 3h. Then it was transferred to an ultrasonic cleaner at 60℃ and ultrasonically extracted for 30min at 200W. The mixture was filtered through a Buchner funnel to obtain the total flavonoid extract.

[0100] (2) The total flavonoid extract prepared in step (1) was distilled under reduced pressure at 45°C and -0.1 MPa until no liquid flowed out. 125 mL of anhydrous ethanol (5 mL / g based on the mass of raw material Hangzhou white chrysanthemum powder) was added, and after thorough shaking, it was transferred to a centrifuge tube and centrifuged at 5000 r / min for 10 min. The supernatant was distilled at 45°C and -0.1 MPa to about 12.5 mL, transferred to a clean petri dish, and dried under vacuum at 50°C and -0.1 MPa to obtain the total flavonoid extract.

[0101] Following the steps above, 2.92g of total flavonoid extract was obtained, with a total flavonoid content of 52.8%, which means 1.54g of total flavonoids were obtained, and the extraction yield was 6.16%.

[0102] Comparing the results of Example 6 and Comparative Example 3, it can be seen that before extracting the total flavonoids from Hangzhou white chrysanthemum, the addition of crude enzyme solution prepared by Aspergillus terrestris fermentation to hydrolyze the flavonoids increased the total flavonoid extraction yield from 6.16% to 7.64%, an increase of 24.0%.

Claims

1. Aspergillus terreus ( Aspergillus terreus HBJ5-32, deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number: GDMCC No: 62493, deposited on May 18, 2022, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province; postcode 510070.

2. The application of Aspergillus terreus HBJ5-32 as described in claim 1 in the extraction of total flavonoids from Hangzhou white chrysanthemum, characterized in that, The application method is as follows: (1) Aspergillus terrestris HBJ5-32 is inoculated onto PDA plate medium and cultured at 28–30℃ for 48–60 h to obtain plate culture; sterile physiological saline is added to the plate culture, and the spores are suspended by stirring with an inoculation loop to obtain Aspergillus terrestris HBJ5-32 spore liquid; Aspergillus terrestris HBJ5-32 spore liquid is inoculated into enzyme production medium at a volume fraction of 6%–8% and cultured at 30℃ and 200–250 r / min for 60–72 h, the fermentation broth is filtered, and the filtrate is the crude enzyme solution; the final concentration composition of the PDA plate medium is: potato 200 g / L, glucose 20 g / L, agar 20 g / L, the solvent is tap water, and the pH is natural; the final concentration composition of the enzyme production medium is: wheat bran 30–40 g / L, (NH4)2SO4 5–6 g / L, KH2PO4 3–5 g / L, MgSO4·7H2O 0.5–1.0 g / L, CaCl2 0.3–0.5 g / L, FeSO4·7H2O 0.1–0.2 g / L, solvent is tap water, pH 6.0–6.5; (2) Mix the crude enzyme solution with Hangzhou white chrysanthemum powder, and enzymatically hydrolyze at 35–40℃ for 4–6 h. Then add anhydrous ethanol to the enzymatic hydrolysis system and perform ultrasonic extraction. Filter to obtain total flavonoid extract; (3) Evaporate the total flavonoid extract under reduced pressure, dissolve in anhydrous ethanol, concentrate and vacuum dry to obtain total flavonoid extract.

3. The application as described in claim 2, characterized in that, The volume of crude enzyme solution used in step (2) is 4–8 mL / g based on the mass of Hangzhou white inulin.

4. The application as described in claim 2, characterized in that, The Hangzhou white chrysanthemum powder mentioned in step (2) is made by drying Hangzhou white chrysanthemum at 85℃ for 2 hours and then pulverizing it and passing it through a 20-mesh sieve.

5. The application as described in claim 2, characterized in that, In step (2), the volume of anhydrous ethanol used is 3-4 times the volume of the crude enzyme solution.

6. The application as described in claim 2, characterized in that, Step (2) The preparation method of total flavonoid extract is as follows: add anhydrous ethanol to the enzymatic hydrolysis system, stir evenly, and then place it in a water bath at 50–60℃ for 2–3 h, and then transfer it to an ultrasonic cleaner at 50–60℃ and 100–200W for 30–60 min; after ultrasonic ethanol extraction, filter while hot to obtain total flavonoid extract.

7. The application as described in claim 2, characterized in that, Step (3) The method for recovering total flavonoid extract from total flavonoid extract is as follows: the total flavonoid extract is distilled at 45℃ and –0.1 MPa until no liquid flows out, anhydrous ethanol is added, and after thorough shaking, it is centrifuged at 5000–8000 r / min for 5–10 min. The supernatant is distilled at 45℃ and –0.1 MPa to 1 / 10 of the original ethanol volume, transferred to a clean petri dish, and vacuum dried at 50℃ and –0.1 MPa to obtain total flavonoid extract; the volume of anhydrous ethanol used is 5–10 mL / g based on the mass of the raw material Hangzhou white chrysanthemum powder.

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